Cisplatin is a main chemotherapeutic drug used to treat non-small-cell lung cancer patients. However, these patients commonly face cisplatin resistance. The roles and underlying mechanisms of gemcitabine, irinotecan, pemetrexed and docetaxel used as single agents or combined with cisplatin for overcoming cisplatin-resistant non-small-cell lung cancer were explored in this study. MTT assays showed that gemcitabine alone exhibited stronger cytotoxicity on cisplatin-resistant A549 cells than irinotecan, pemetrexed and docetaxel. Meanwhile, gemcitabine combined with cisplatin showed a synergistic inhibitory effect on cisplatin-resistant cells. RNA sequencing and Gene Ontology/Kyoto Encyclopedia of Genes and Genomes analysis showed that cell cycle signaling pathways and trx-interacting protein were factors in the efficacy of the cotreatment. Flow cytometry and Western blot results showed that when cisplatin-resistant A549 cells were cotreated with gemcitabine and cisplatin, G0/G1 phase arrest occurred, and trx-interacting protein was upregulated. Silencing trx-interacting protein attenuated the response of the resistant cells to the drug combination. A trx-interacting protein agonist together with cisplatin showed an additive cytotoxic effect on the resistant cells compared with cisplatin alone. The gemcitabine and cisplatin combination, compared to gemcitabine or PBS alone, markedly suppressed the growth of cisplatin-resistant A549 tumors in vivo, accompanied by an increase in trx-interacting protein and a decrease in Ki67 expression. Therefore, we concluded that gemcitabine and cisplatin, as an FDA-approved combination, is a viable therapy for cisplatin-resistant non-small-cell lung cancer ex vivo and in vivo. (C) 2020 Elsevier Inc. All rights reserved.